Adjuvanted formulations of booster vaccines

ABSTRACT

The invention improves TdaP vaccines by including a TLR agonist in them. This agonist can provide stronger protection, longer-lasting protection, and/or can reduce the amount of antigen which is required to achieve a particular immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Division of U.S. patent application Ser. No. 15/163,592, filed May 24, 2016, patented as 9,931,399 on Apr. 3, 2018, which is a continuation of U.S. patent application Ser. No. 13/790,948, filed Mar. 8, 2013, patented as U.S. Pat. No. 9,375,471 on Jun. 28, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 61/608,398, filed Mar. 8, 2012, and 61/697,730, filed Sep. 6, 2012, all of which are hereby expressly incorporated by reference into the present application.

TECHNICAL FIELD The invention is in the field of booster vaccines for diphtheria, tetanus and pertussis. BACKGROUND ART

Two adolescent DTP booster vaccines are currently available—BOOSTRIX™ & ADACEL™ [1]. Both vaccines contain diphtheria toxoid, tetanus toxoid and acellular pertussis antigens. They are also available in combination with inactivated poliovirus (BOOSTRIX POLIO™ and ADACEL POLIO™). All of these vaccines include an aluminium salt adjuvant.

In general these vaccines are known as TdaP vaccines, in contrast to pediatric DTaP vaccines. A common feature is that, relative to their pediatric counterparts, they have lower antigen doses e.g. the diphtheria toxoid content of BOOSTRIX™ is 10-fold lower than in the INFANRIX™ products, and in ADACEL™ it is 7.5-fold lower than in DAPTACEL™. Moreover, the ratio of antigenic components is also altered. In particular, the ratio of diphtheria and tetanus toxoids is 2.5:1 in the INFANRIX™ products but is 1:2 in the BOOSTRIX™ product, and is 3:1 in the DAPTACEL™ product but is 1:2.5 in the ADACEL™ product. Thus these booster vaccines show a large reduction in the dose of diphtheria toxoid, both in absolute amounts and also relative to the tetanus toxoid content. Some of the pertussis components also differ from the levels seen in the pediatric counterparts, but the levels of the poliovirus antigens are the same in both the pediatric and adolescent vaccines. Based on public information the compositions are as follows:

D T Pa⁽¹⁾ IPV⁽³⁾ Volume Al⁺⁺⁺ Boostrix ™ 2.5 Lf 5 Lf 8/8/2.5 — 0.5 ml ≤0.39 mg Boostrix 2.5 Lf 5 Lf 8/8/2.5 40/8/32 0.5 ml  0.5 mg Polio ™ Adacel ™   2 Lf 5 Lf 2.5/5/3⁽²⁾ — 0.5 ml  0.33 mg Adacel   2 Lf 5 Lf 2.5/5/3⁽²⁾ 40/8/32 0.5 ml  0.33 mg Polio ™ Notes: ⁽¹⁾Pa dose shows amounts of pertussis toxoid, then FHA, then pertactin (μg). ⁽²⁾Adacel's Pa components also contain 5 μg fimbriae types 2 and 3. ⁽³⁾IPV dose shows amounts of type 1, then type 2, then type 3 (measured in DU).

It is an object of the invention to provide further and improved TdaP vaccines suitable for human use as a booster in adults, adolescents and children aged four years and older who have previously received childhood immunisation.

DISCLOSURE OF THE INVENTION

In a first aspect, the invention improves current TdaP vaccines by including a TLR agonist in them. This agonist can provide stronger protection, longer-lasting protection, and/or can reduce the amount of antigen which is required to achieve a particular immune response.

In a second aspect, the invention improves current TdaP vaccines by adjuvanting them with an oil-in-water emulsion. This emulsion can again improve protection relative to known TdaP vaccines.

For the first aspect the invention therefore provides an immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, an aluminium salt adjuvant, and a TLR agonist, wherein the composition includes an excess of tetanus toxoid relative to diphtheria toxoid (in Lf units).

The TLR agonist is ideally a TLR4 agonist or a TLR7 agonist. Preferably, the TLR agonist and/or at least one of the toxoids is/are adsorbed to the aluminium salt adjuvant.

By including a TLR agonist it is possible for the compositions to have a lower amount of antigen and/or lower amount of aluminium relative to known vaccines, while nevertheless having comparable immunogenicity.

Ideally the composition has one or more of the following properties:

-   -   an Al⁺⁺⁺ concentration of ≤0.5 mg/ml;     -   a diphtheria toxoid concentration of ≤4 Lf/ml;     -   a tetanus toxoid concentration of ≤9 Lf/ml; and/or     -   a pertussis toxoid concentration of ≤4 μg/ml.

For instance, where the immunogenic composition is in a unit dose form for administration to a patient (e.g. in a 0.5 ml volume), it can have one or more of the following properties:

-   -   an Al⁺⁺⁺ content of ≤0.255 mg;     -   a diphtheria toxoid content of ≤2 Lf;     -   a tetanus toxoid content of ≤4.5 Lf; and/or     -   a pertussis toxoid content of ≤2 μg.

For the second aspect the invention provides an immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and an oil-in-water emulsion adjuvant, wherein the composition includes an excess of tetanus toxoid relative to diphtheria toxoid (in Lf units).

Ideally the composition has one or more of the following properties:

-   -   a diphtheria toxoid concentration of ≤4 Lf/ml;     -   a tetanus toxoid concentration of ≤9 Lf/ml; and/or     -   a pertussis toxoid concentration of ≤4 μg/ml.

For instance, where the immunogenic composition is in a unit dose form for administration to a patient (e.g. in a 0.5 ml volume), it can have one or more of the following properties:

-   -   a diphtheria toxoid content of ≤2 Lf;     -   a tetanus toxoid content of ≤4.5 Lf; and/or     -   a pertussis toxoid content of ≤2 μg.

For both aspects, compositions of the invention can include antigens in addition to diphtheria toxoid, tetanus toxoid, and pertussis toxoid e.g. they can include an inactivated poliovirus (IPV) component.

Aluminium Salts

TLR agonists can adsorb to insoluble aluminium salts to form an adsorbed complex for adjuvanting TdaP immunogens. Such aluminium salts have a long history of use in vaccines.

Useful aluminium salts include, but are not limited to, aluminium hydroxide and aluminium phosphate adjuvants. Such salts are described e.g. in chapters 8 & 9 of reference 2, and chapter 4 of reference 3. Aluminium salts which include hydroxide ions are preferred for use with the present invention as these hydroxide ions can readily undergo ligand exchange. Thus preferred salts for adsorption of TLR agonists are aluminium hydroxide and/or aluminium hydroxyphosphate. These have surface hydroxyl moieties which can readily undergo ligand exchange with phosphorus-containing groups (e.g. phosphates, phosphonates) to provide stable adsorption. An aluminium hydroxide adjuvant is most preferred.

The adjuvants commonly known as “aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm⁻¹ and a strong shoulder at 3090-3100cm⁻¹ (chapter 9 of ref. 2). The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants e.g. with needle-like particles with diameters about 2 nm. The pI of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants commonly known as “aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO₄ by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.) indicates the presence of structural hydroxyls (chapter 9 of reference 2).

The PO₄/Al⁺⁺⁺ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95±0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, included at 0.6 mg Al⁺⁺⁺/ml. The aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs, with primary particles in the range of 50 nm). Typical diameters of the particles are in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigen adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

In solution both aluminium phosphate and hydroxide adjuvants tend to form stable porous aggregates 1-10 μm in diameter [4].

A composition including a TLR agonist adsorbed to an aluminium salt can also include a buffer (e.g. a phosphate or a histidine or a Tris buffer). When such a composition includes a phosphate buffer, however, it is preferred that the concentration of phosphate ions in the buffer should be less than 50 mM e.g. <40 mM, <30 mM, <20 mM, <10 mM, or <5 mM, or between 1-15 mM. A histidine buffer is preferred e.g. between 1-50 mM, between 5-25 mM, or about 10 mM.

Because of the insolubility of adsorptive aluminium salts which are useful with the invention, compositions containing adsorbed TLR agonists will generally be suspensions having a cloudy appearance. This can mask contaminating bacterial growth and so a composition of the invention may include a preservative such as thiomersal or 2-phenoxyethanol. It is preferred that a composition should be substantially free from (e.g. <10 μg/ml) mercurial material e.g. thiomersal-free. Compositions containing no mercury are more preferred. Preservative-free compositions are also possible

A composition can include a mixture of both an aluminium hydroxide and an aluminium phosphate, and a TLR agonist may be adsorbed to one or both of these salts.

The concentration of Al⁺⁺⁺ in a composition for administration to a patient is preferably less than 0.5 mg/ml e.g. ≤0.4 mg/ml, ≤0.3 mg/ml, ≤0.2 mg/ml, ≤0.1 mg/ml, etc. Because the inclusion of a TLR agonist can improve the adjuvant effect of aluminium salts then the invention advantageously permits lower amounts of Al⁺⁺⁺ per dose, and so a composition of the invention can usefully include between 10 and 250 μg of Al⁺⁺ per unit dose. Current TdaP vaccines include at least 330 μg Al⁺⁺⁺ per dose. In concentration terms, a composition of the invention may have an Al⁺⁺⁺ concentration between 10 and 500 μg/ml e.g. between 10-300 μg/ml, between 10-200 μg/ml, or between 10-100 μg/ml.

In general, the weight ratio of TLR agonist to Al⁺⁺⁺ will be less than 5:1 e.g. less than 4:1, less than 3:1, less than 2:1, or less than 1:1. Thus, for example, with an Al⁺⁺⁺ concentration of 0.5 mg/ml the maximum concentration of TLR agonist would be 2.5 mg/ml. But higher or lower levels can be used. A lower mass of TLR agonist than of Al⁺⁺⁺ can be most typical e.g. per dose, 100 μg of TLR agonist with 0.2 mg Al⁺⁺⁺, etc. For instance, the Fendrix product includes 50 μg of 3d-MPL and 0.5 mg Al⁺⁺⁺ per dose.

It is preferred that at least 50% (by mass) of TLR agonist(s) in the composition is adsorbed to the aluminium salt e.g. ≥60%, ≥70%, ≥80%, ≥85%, ≥90%, ≥92%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or even 100%.

TLR Agonists

In its first aspect, compositions of the invention include a TLR agonist i.e. a compound which can agonise a Toll-like receptor. Most preferably, a TLR agonist is an agonist of a human TLR. The TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11; preferably it can activate human TLR4 or human TLR7.

Agonist activity of a compound against any particular Toll-like receptor can be determined by standard assays. Companies such as Imgenex and Invivogen supply cell lines which are stably co-transfected with human TLR genes and NFκB, plus suitable reporter genes, for measuring TLR activation pathways. They are designed for sensitivity, broad working range dynamics and can be used for high-throughput screening. Constitutive expression of one or two specific TLRs is typical in such cell lines. See also reference 5. Many TLR agonists are known in the art e.g. reference 6 describes certain lipopeptide molecules that are TLR2 agonists, references 7 to 10 each describe classes of small molecule agonists of TLR7, and references 11 & 12 describe TLR7 and TLR8 agonists for treatment of diseases.

A TLR agonist used with the invention ideally includes at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour [13].

Phosphorus-containing adsorptive moieties are particularly useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc.

Preferably the TLR agonist includes at least one phosphonate group.

Thus, in preferred embodiments, a composition of the invention includes a TLR agonist (more preferably a TLR7 agonist) which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt [13].

TLR agonists useful with the invention may include a single adsorptive moiety, or may include more than one e.g. between 2 and 15 adsorptive moieties. Typically a compound will include 1, 2 or 3 adsorptive moieties.

Phosphorus-containing TLR agonists useful with the invention can be represented by formula (A1):

wherein:

-   -   R^(X) and R^(Y) are independently selected from H and C₁-C₆         alkyl;     -   X is selected from a covalent bond, O and NH;     -   Y is selected from a covalent bond, O, C(O), S and NH;     -   L is a linker e.g. selected from, C₁-C₆alkylene,         C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and         —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1         to 4 substituents independently selected from halo, OH,         C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6;     -   q is selected from 1, 2, 3 and 4;     -   n is selected from 1, 2 and 3; and     -   A is a TLR agonist moiety.

In one embodiment, the TLR agonist according to formula (A1) is as follows: R^(X) and R^(Y) are H; X is O; L is selected from C₁-C₆ alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selected from 1 and 2; and n is 1. Thus in these embodiments the adsorptive moiety comprises a phosphate group.

In other embodiments, the TLR agonist according to formula (A1) is as follows: R^(X) and R^(Y) are H; X is a covalent bond; L is selected from C₁-C₆ alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)' each optionally substituted with 1 to 2 halogen atoms; p is selected from 1, 2 or 3; q is selected from 1 or 2; and n is 1. Thus in these embodiments the adsorptive moiety comprises a phosphonate group.

Useful ‘A’ moieties for formula (A1) include, but are not limited to, radicals of any of the following compounds, defined herein or as disclosed in references 4-13 and 14-48:

In some embodiments, the TLR agonist moiety ‘A’ has a molecular weight of less than 1000 Da. In some embodiments, the TLR agonist of formula (A1) has a molecular weight of less than 1000 Da.

Preferred TLR agonists are water-soluble. Thus they can form a homogenous solution when mixed in an aqueous buffer with water at pH 7 at 25° C. and 1 atmosphere pressure to give a solution which has a concentration of at least 50 μg/ml. The term “water-soluble” thus excludes substances that are only sparingly soluble under these conditions.

Useful TLR agonists include those having formula (C), (D), (E), (F), (G), (H), (I), (II), (J) or (K) as described in more detail below. Other useful TLR agonists are compounds 1 to 102 as defined in reference 13. Preferred TLR7 agonists have formula (K), such as compound K2 identified below. These can be used as salts e.g. the arginine salt of K2.

Preferred TLR4 agonists are analogs of monophosphoryl lipid A (MPL), as described in more detail below. For instance, a useful TLR4 agonist is a 3d-MPL.

A composition of the invention can include more than one TLR agonist. These two agonists are different from each other and they can target the same TLR or different TLRs. Both agonists can be adsorbed to an aluminium salt.

Formulae (C), (D), (E) and (H) TLR7 Agonists

The TLR agonist can be a compound according to any of formulae (C), (D), (E), and (H):

wherein:

-   -   (a) P³ is selected from H, C₁-C₆alkyl, CF₃, and         —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and         -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁴ is selected from H,         C₁-C₆alkyl, —C₁-C₆alkylaryl and -Y-L-X—P(O)(OR^(X))(OR^(Y));         with the proviso that at least one of P³ and P⁴ is         -Y-L-X—P(O)(OR^(X))(OR^(Y)),     -   (b) P⁵ is selected from H, C₁-C₆alkyl, and         -Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁶ is selected from H, C₁-C₆alkyl         each optionally substituted with 1 to 3 substituents selected         from C₁-C₄alkyl and OH, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷         is selected from H, C₁-C₆alkyl,         —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)—, —NHC₁-C₆alkyl and         -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one         of P⁵, P⁶ and P⁷ is -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   (c) P⁸ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,         —NHC₁-C₆alkyl each optionally substituted with OH, and         -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are each         independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,         —NHC₁-C₆alkyl each optionally substituted with OH and         C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso         that at least one of P⁸, P⁹ or P¹⁰ is         -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   (d) P¹⁶ and each P¹⁸ are each independently selected from H,         C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected         from H, C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl         heteroaryl, C₁-C₆alkylaryl-Y-L-X—P(O)(OR^(X))(OR^(Y)) and         -Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1         to 2 substituents selected from C₁-C₆alkyl or heterocyclyl with         the proviso that at least one of P¹⁶, P¹⁷ or a P¹⁸ contains a         -Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety;     -   R^(X) and R^(Y) are independently selected from H and         C₁-C₆alkyl;     -   R^(C), R^(D) and R^(H) are each independently selected from H         and C₁-C₆alkyl;     -   X^(C) is selected from CH and N;     -   R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,         C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—;     -   X^(E) is selected from a covalent bond, CR^(E2)R^(E3) and         NR^(E4);     -   R^(E2), R^(E3) and R^(E4) are independently selected from H and         C₁-C₆alkyl;     -   X^(H1)—X^(H2) is selected from —CR^(H2)R^(H3)—,         —CR^(H2)R^(H3)—CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—,         —C(O)CR^(H2)R^(H3)—, —CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—,         C(O)NR^(H4)—, CR^(H2)R^(H3)S(O)₂ and —CR^(H2)═CR^(H2)—;     -   R^(H2), R^(H3) and R^(H4) are each independently selected from         H, C₁-C₆alkyl and P¹⁸;     -   X^(H3) is selected from N and CN;     -   X is selected from a covalent bond, O and NH;     -   Y is selected from a covalent bond, O, C(O), S and NH;     -   L is selected from, a covalent bond C₁-C₆alkylene,         C₁-C₆alkenylene, arylene, heteroarylene,     -   C₁-C₆alkyleneoxy and —((CH₂)_(p)O)₉(CH₂)_(p)— each optionally         substituted with 1 to 4 substituents independently selected from         halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; m is selected         from 0 or 1;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6;     -   q is selected from 1, 2, 3 and 4; and     -   s is selected from 0 and 1.         Formula (G)—TLR8 Agonist

The TLR agonist can be a compound according to formula (G):

wherein:

-   -   P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆ alkoxy, NR^(V)R^(W)         and -Y-L-X—P(O)(OR^(C))(OR^(Y));     -   P¹² is selected from H, C₁-C₆alkyl, aryl optionally substituted         by —C(O)NR^(V)R^(W), and -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   P¹³, P¹⁴ and P¹⁵ are independently selected from H, C₁-C₆alkyl,         C₁-C₆ alkoxy and -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   with the proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ or P¹⁵         is -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   R^(V) and R^(W) are independently selected from H, C₁-C₆alkyl or         together with the nitrogen atom to which they are attached form         a 4 to 7 remembered heterocyclic ring;     -   X^(G) is selected from C, CH and N;     -   represents an optional double bond, wherein X^(G) is C if         is a double bond; and     -   R^(G) is selected from H and C₁-C₆alkyl;     -   X is selected from a covalent bond, O and NH;     -   Y is selected from a covalent bond, O, C(O), S and NH;     -   L is selected from, a covalent bond C₁-C₆alkylene,         C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and         —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1         to 4 substituents independently selected from halo, OH,         C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6 and     -   q is selected from 1, 2, 3 and 4.         Formulae (I) and (II)—TLR 7 agonists [8]

The TLR agonist can be a compound according to formula (I) or formula (II):

wherein:

-   -   Z is —NH₂ or —OH;     -   X¹ is alkylene, substituted alkylene, alkenylene, substituted         alkenylene, alkynylene, substituted alkynylene, carbocyclylene,         substituted carbocyclylene, heterocyclylene, or substituted         heterocyclylene;     -   L¹ is a covalent bond, arylene, substituted arylene,         heterocyclylene, substituted heterocyclylene, carbocyclylene,         substituted carbocyclylene, —S—, —S(O)—, S(O)₂, —NR⁵—, or —O—     -   X² is a covalent bond, alkylene, or substituted alkylene;     -   L² is NR⁵—, —N(R⁵)C(O)—, —O—, —S—, —S(O)—, S(O)₂, or a covalent         bond;     -   R³ is H, alkyl, substituted alkyl, heteroalkyl, substituted         heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted         aryl, arylalkyl, substituted arylalkyl, heterocyclyl,         substituted heterocyclyl, heterocyclylalkyl, or substituted         heterocyclylalkyl;     -   Y¹ and Y² are each independently a covalent bond, —O— or —NR⁵—;         or —Y¹ —R¹ and —Y²—R² are each independently —O—N═C(R⁶R⁷);     -   R¹ and R² are each independently H, alkyl, substituted alkyl,         carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted         heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted         alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl,         substituted heterocyclylalkyl, -alkylene-C(O)—O—R⁵, (substituted         alkylene)-C(O)—O—R⁵, -alkylene-O—C(O)—R⁵, -(substituted         alkylene)-O—C(O)—R⁵, -alkylene-O—C(O)—O—R⁵, or -(substituted         alkylene)-O—C(O)—O—R⁵     -   R⁴ is H, halogen, —OH, —O-alkyl, —O-alkylene-O—C(O)—O—R⁵, —O—C(O         )—O—R⁵, —SH, or —NH(R⁵);     -   each R⁵, R⁶, and R⁷ are independently H, alkyl, substituted         alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl,         substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl,         substituted alkynyl, arylalkyl, substituted arylalkyl,         heterocyclylalkyl, or substituted heterocyclylalkyl.         Formula (J)—TLR2 Agonists [14]

The TLR agonist can be a compound according to formula (J):

wherein:

-   -   R¹ is H, —C(O)—C₇-C₁₈alkyl or —C(O)—C₁-C₆alkyl;     -   R² is C₇-C₁₈alkyl;     -   R³ is C₇-C₁₈alkyl;     -   L₁ is —CH₂OC(O)—, —CH₂O—, —CH₂NR⁷C(O)— or —CH₂OC(O)NR⁷—;     -   L₂ is —O—OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—;     -   R⁴ is -L₃R⁵ or -L₄R⁵;     -   R⁵ is —(R⁷)₂, —OR⁷, —P(O)(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸,         —NR⁷C(O)L₄R⁸, —OL₃R⁶, —C(O)NR⁷L₃R⁸, —C(O)NR⁷L₄R⁸, —S(O)₂OR⁷,         —OS(O)₂OR⁷, C₁-C₆alkyl, a C₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14         ring membered heteroaryl containing 1 to 3 heteroatoms selected         from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 ring membered         heterocycloalkyl containing 1 to 3 heteroatoms selected from O,         S and N, wherein the aryl, heteroaryl, cycloalkyl and         heterocycloalkyl of R⁵ are each unsubstituted or the aryl,         heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are each         substituted with 1 to 3 substituents independently selected from         —OR⁹, —OL₃R⁶, —OL₄R⁶, —OR⁷, and —C(O)OR⁷;     -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is         unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1         to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with         2 C₁-C₆alkyl groups on the same carbon atom which together,         along with the carbon atom they are attached to, form a         C₃-C₈cycloakyl;     -   L₄ is —((CR7R7)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or         —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p), wherein each R¹¹ is a         C₁-C₆alkyl groups which together, along with the carbon atom         they are attached to, form a C₃-C₈cycloakyl;     -   each R⁶ is independently selected from halo, C₁-C₆alkyl,         C₁-C₆alkyl substituted with 1-2 hydroxyl groups, —OR⁷, —N(R⁷)₂,         —C(O)OH, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀aryl and a         C₁₄aryl;     -   each R⁷ is independently selected from H and C₁-C₆alkyl;     -   R⁸ is selected from —SR⁷, —C(O )OH, —P(O)(OR⁷)₂, and a 5 to 6         ring membered heterocycloalkyl containing 1 to 3 heteroatoms         selected from O and N;     -   R⁹ is phenyl;     -   each R¹⁰ is independently selected from H and halo;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and     -   q is 1, 2, 3 or 4.

Preferably R⁵ is P(O)(OR⁷)₂, —NR⁷C(O)L₃-P(O)(OR⁷)₂, —NR⁷C(O)L₄-P(O)(OR⁷)₂, —OL₃-P(O)(OR⁷)₂, —C(O)NR⁷L₃-P(O)(OR⁷)₂, or —C(O)NR⁷L₄-P(O)(OR⁷)₂.

In some embodiments of (J), R₁ is H. In other embodiments of (J), R₁ is —C(O)—C₁₅alkyl;

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—, —O—, —NR⁷C(O)— or —C(O )NR⁷—; or (ii) or L₁ is —CH₂O— and L₂ is —OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—; or (iii) L₁ is —CH₂NR⁷C(O)— and L₂ is —OC(O)—, —NR⁷C(O)— or —OC(O )NR⁷—; or (iv) L₁ is —CH₂OC(O)NR⁷— and L₂ is —OC(O)—, —O—, NR⁷C(O)— or —OC(O)NR⁷—.

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—; or (ii) L₁ is —CH₂O— and L₂ is —O—; or (iii) L₁ is —CH₂O— and L₂ is —NHC(O)—; or (iv) L₁ is —CH₂OC(O)NH— and L₂ is —OC(O)NH—.

In some embodiments of (J), (i) R² is —C₁₁alkyl and R³ is —C₁₁alkyl; or (ii) R² is —C₁₆alkyl and R³ is —C₁₆alkyl; or (iii) R² is —C₁₆alkyl and R³ is —C₁₁alkyl; or (iv) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (v) R² is —C₇alkyl and R³ is —C₇alkyl; or (vi) R² is —C₉alkyl and R³ is —C₉alkyl; or (vii) R² is —C₈alkyl and R³ is —C₈alkyl; or (viii) R² is —C₁₃alkyl and R³ is —C₁₃alkyl; or (ix) R² is —C₁₂alkyl and R³ is —C₁₁alkyl; or (x) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (xi) R² is —C₁₀alkyl and R³ is —C₁₀alkyl; or (xii) R² is —C₁₅alkyl and R³ is —C₁₅alkyl.

In some embodiments of (J), R² is —C₁₁alkyl and R³ is —C₁₁alkyl.

In some embodiments of (J), L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is unsubstituted or is substituted with 1 to 4 R⁶ groups.

In some embodiments of (J): L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—; each R¹⁰ is independently selected from H and F; and each p is independently selected from 2, 3, and 4.

In some embodiments of (J), each R⁶ is independently selected from methyl, ethyl, i-propyl, i-butyl, —CH₂OH, —OH, —F, —NH₂, —C(O )OH, —C(O)NH₂, —P(O)(OH)₂ and phenyl.

In some embodiments of (J), each R⁷ is independently selected from H, methyl and ethyl.

TLR4 Agonists

Compositions of the invention can include a TLR4 agonist, and most preferably an agonist of human TLR4. TLR4 is expressed by cells of the innate immune system, including conventional dendritic cells and macrophages [15]. Triggering via TLR4 induces a signalling cascade that utilizes both the MyD88- and TRIF-dependent pathways, leading to NF-κB and IRF3/7 activation, respectively.

TLR4 activation typically induces robust IL-12p70 production and strongly enhances Th1-type cellular and humoral immune responses.

Various useful TLR4 agonists are known in the art, many of which are analogs of endotoxin or lipopolysaccharide (LPS). For instance, the TLR4 agonist can be:

-   -   (i) 3d-MPL (i.e. 3-O-deacylated monophosphoryl lipid A; also         known as 3-de-O-acylated monophosphoryl lipid A or         3-O-desacyl-4′-monophosphoryl lipid A). This derivative of the         monophosphoryl lipid A portion of endotoxin has a de-acylated         position 3 of the reducing end of glucosamine. It has been         prepared from a heptoseless mutant of Salmonella minnesota, and         is chemically similar to lipid A but lacks an acid-labile         phosphoryl group and a base-labile acyl group. Preparation of         3d-MPL was originally described in ref. 16, and the product has         been manufactured and sold by Corixa Corporation. It is present         in GSK's ‘AS04’ adjuvant. Further details can be found in         references 17 to 20.     -   (ii) glucopyranosyl lipid A (GLA) [21] or its ammonium salt:

-   -   (iii) an aminoalkyl glucosaminide phosphate, such as RC-529 or         CRX-524 [22-24]. RC-529 and CRX-524 have the following         structure, differing by their R₂ groups:

-   -   (iv) compounds containing lipids linked to a         phosphate-containing acyclic backbone, such as the TLR4         antagonist E5564 [25,26]:

-   -   (v) A compound of formula I, II or III as defined in reference         27, or a salt thereof, such as compounds ‘ER 803058’, ‘ER         803732’, ‘ER 804053’, ‘ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER         804680’, ‘ER 803022’, ‘ER 804764’ or ‘ER 804057’. ER 804057 is         also known as E6020 and it has the following structure:

-   -   whereas ER 803022 has the following structure:

-   -   (vi) One of the polypeptide ligands disclosed in reference 28.

Any of these TLR4 agonists can be used with the invention.

A composition of the invention can include an aluminium salt to which the TLR4 agonist is adsorbed. TLR4 agonists with adsorptive properties typically include a phosphorus-containing moiety which can undergo ligand exchange with surface groups on an aluminium salt, and particularly with a salt having surface hydroxide groups. Thus a useful TLR4 agonist may include a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, a phosphate, etc. Preferred TLR4 agonists include at least one phosphate group [13] e.g. the agonists (i) to (v) listed above.

The preferred TLR4 agonist for use with the invention is 3d-MPL. This can be adsorbed to an aluminium phosphate adjuvant, to an aluminium hydroxide adjuvant, or to a mixture of both [29].

3d-MPL can take the form of a mixture of related molecules, varying by their acylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be of different lengths). The two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their 2-position carbons (i.e. at positions 2 and 2′), and there is also O-acylation at the 3′ position. The group attached to carbon 2 has formula —NH—CO—CH₂—CR¹R^(1′). The group attached to carbon 2′ has formula —NH—CO—CH₂—CR²R^(2′). The group attached to carbon 3′ has formula —O—CO—CH₂—CR³R^(3′). A representative structure is:

Groups R¹, R² and R³ are each independently —(CH₂)—CH₃. The value of n is preferably between 8 and 16, more preferably between 9 and 12, and is most preferably 10.

Groups R^(1′), R^(2′) and R^(3′) can each independently be: (a) —H; (b) —OH; or (c) —O—CO—R⁴, where R⁴ is either —H or —(CH₂)_(m)—CH₃, wherein the value of m is preferably between 8 and 16, and is more preferably 10, 12 or 14. At the 2 position, m is preferably 14. At the 2′ position, m is preferably 10. At the 3′ position, m is preferably 12. Groups R^(1′), R^(2′) and R^(3′) are thus preferably —O-acyl groups from dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

When all of R^(1′), R^(2′) and R^(3′) are H then the 3d-MPL has only 3 acyl chains (one on each of positions 2, 2′ and 3′). When only two of R^(1′), R^(2′) and R^(3′) are —H then the 3d-MPL can have 4 acyl chains. When only one of R^(1′), R^(2′) and R^(3′) is—H then the 3d-MPL can have 5 acyl chains. When none of R^(1′), R^(2′) and R^(3′) is —H then the 3d-MPL can have 6 acyl chains. The 3d-MPL used according to the invention can be a mixture of these forms, with from 3 to 6 acyl chains, but it is preferred to include 3d-MPL with 6 acyl chains in the mixture, and in particular to ensure that the 6 acyl chain form makes up at least 10% by weight of the total 3d-MPL e.g. ≥20%, ≥30%, ≥40%, ≥50% or more. 3d-MPL with 6 acyl chains has been found to be the most adjuvant-active form.

Thus the most preferred form of 3d-MPL for use with the invention is:

Where 3d-MPL is used in the form of a mixture then references to amounts or concentrations of 3d-MPL in compositions of the invention refer to the combined 3d-MPL species in the mixture.

Typical compositions include 3d-MPL at a concentration of between 25 μg/ml and 200 μg/ml e.g. in the range 50-150 μg/ml, 75-125 μg/ml, 90-110 μg/ml, or about 100 μg/ml. It is usual to administer between 25-75 μg of 3d-MPL per dose e.g. between 45-55 μg, or about 50 μg 3d-MPL per dose.

In aqueous conditions, 3d-MPL can form micellar aggregates or particles with different sizes e.g. with a diameter <150 nm or >500 nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g. small enough to give a clear aqueous suspension of 3d-MPL) are preferred for use according to the invention because of their superior activity [30]. Preferred particles have a mean diameter less than 150 nm, more preferably less than 120nm, and can even have a mean diameter less than 100 nm. In most cases, however, the mean diameter will not be lower than 50 nm. Where 3d-MPL is adsorbed to an aluminum salt then it may not be possible to measure the 3D-MPL particle size directly, but particle size can be measured before adsorption takes place. Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g. ≥60%, ≥70%, ≥80%, ≥90%, or more) of the particles will have a diameter within the range x±25%.

Formula (K) [31]

The TLR agonist can be a compound according to formula (K):

wherein:

-   -   R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, -L¹R⁵, -L¹R⁶ _(, -L) ²R⁵, -L²R⁶,         or —OL²R⁶;     -   L¹ is —C(O)—or —O—;     -   L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or         —((CR⁴R⁴)_(p)O)₈(CH₂)_(p), wherein the C₁-C₆alkylene and         C₂-C₆alkenylene of L² are optionally substituted with 1 to 4         fluoro groups;     -   each L³ is independently selected from C₁-C₆alkylene and         —((CR⁴R⁴)_(p)O)₈(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³ is         optionally substituted with 1 to 4 fluoro groups;     -   L⁴ is arylene or heteroarylene;     -   R² is H or C₁-C₆alkyl;     -   R³ is selected from C₁-C₄alkyl, -L³R⁵, -L¹R⁵, -L³R⁷, -L³L⁴L³R⁷,         L³L⁴R⁵, -L³L⁴L³R³, —OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR⁸,         —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH;     -   each R⁴ is independently selected from H and fluoro;     -   R⁵ is —P(O)(OR⁹)₂,     -   R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;     -   R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;     -   R⁸ is H or C₁-C₄alkyl;     -   each R⁹ is independently selected from H and C₁-C₆alkyl;     -   R¹⁰ is H or C₁-C₄alkyl;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and     -   q is 1, 2, 3 or 4.

The compound of formula (K) is preferably of formula (K′):

wherein:

-   -   P¹ is selected from H, C₁-C₆alkyl optionally substituted with         COOH and -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   P² is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy and         -Y-L-X—P(O)(OR^(Xx))(OR^(Y));     -   with the proviso that at least one of P¹ and P² is         -Y-L-X—P(O)(OR^(X))(OR^(Y));     -   R^(B) is selected from H and C₁-C₆alkyl;     -   R^(X) and R^(Y) are independently selected from H and         C₁-C₆alkyl;     -   X is selected from a covalent bond, O and NH;     -   Y is selected from a covalent bond, O, C(O), S and NH;     -   L is selected from, a covalent bond C₁-C₆alkylene,         C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and         —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1         to 4 substituents independently selected from halo, OH,         C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;     -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and     -   q is selected from 1, 2, 3 and 4.

In some embodiments of formula (K′): P¹ is selected from C₁-C₆alkyl optionally substituted with COOH and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P² is selected from C₁-C₆alkoxy and -Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(B) is C₁-C₆alkyl; X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.

Formula (F)—TLR7 Agonists [9]

The TLR agonist can be a compound according to formula (F):

wherein:

-   -   X³ is N;     -   X⁴ is N or CR³     -   X⁵ is —CR⁴═CR⁵—;     -   R¹ and R² are H;     -   R³ is H;     -   R⁴ and R⁵ are each independently selected from H, halogen,         —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂,         —NHN(R⁹)₂, —SR⁷, —(CH₂),OR⁷, —(CH₂)_(n)R⁷, - LR⁸, -LR¹⁰, —OLR⁸,         —OLR¹⁰, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,         C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl,         heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein         the C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,         C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,         C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ and R⁵         are each optionally substituted with 1 to 3 substituents         independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,         —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,         —P(O)(OR¹⁹)₂, —OP(O)(OR¹⁹)₂, —C(O)N(R⁹)₇, —S(O)₂R⁸, —S(O)R⁸,         —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;     -   or, R³ and R⁴, or R⁴ and R⁵, or R⁵ and R⁶, when present on         adjacent ring atoms, can optionally be linked together to form a         5-6 membered ring, wherein the 5-6 membered ring is optionally         substituted with R⁷;     -   each L is independently selected from a bond,         —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and         C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and         C₂-C₆alkynylene of L are each optionally substituted with 1 to 4         substituents independently selected from halogen, —R⁸, —OR⁸,         —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and)         —OP(O)(OR¹⁰)₂;     -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,         C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,         C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and         C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl,         C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene,         C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and         C₃-C₈heterocycloalkyl groups of R⁷ are each optionally         substituted with 1 to 3 R¹³ groups, and each R¹³ is         independently selected from halogen, —CN, -LR⁹, -LOR⁹, —OLR⁹,         -LR¹⁰, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸, -LSR⁸, -LSR¹⁰,         -LC(O)R⁸, —OLC(O)R⁸, -LC(O)OR⁸, -LC(O)R¹⁰, -LOC(O)OR⁸,         -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸, -LNR⁹R¹° ,         -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH, -LNR⁹C(O)R⁸,         -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂, -LNR⁹S(O)₂R⁸,         -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂, -LP(O)(OR¹⁰)₂ and         -OLP(O)(OR¹⁰)₂;     -   each R⁸ is independently selected from H, —CH(R¹⁰)₂, C₁-C₈alkyl,         C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy,         C_(1I)-C₆heteroalkyl, C₃-C₈cycloalkyl, C₈heterocycloalkyl,         C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl,         C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,         C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl,         C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each         optionally substituted with 1 to 3 substituents independently         selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,         —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹¹, —NR¹¹R¹¹, R¹²,         —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,         —S(O)R¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and         —OP(O)(OR¹¹)₂;     -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,         —C(O)R¹¹, —C(O)OR¹¹, —S(O)₂R¹¹, —C₁-C₆ alkyl, C₁-C₆ heteroalkyl         and C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆aIkyl         that together with N they are attached to form a         C₃-C₈heterocycloalkyl, wherein the C₃-C₈heterocycloalkyl ring         optionally contains an additional heteroatom selected from N, O         and S, and wherein the C₁-C₅ alkyl, C₁-C₆ heteroalkyl, C₃-C₆         cycloalkyl, or C₃-C₈heterocycloalkyl groups of R⁹ are each         optionally substituted with 1 to 3 substituents independently         selected from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, OC(O)R¹¹,         —C(O)OR¹¹, —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,         —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂ and         —OP(O)(OR¹¹)₂;     -   each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl,         C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl,         C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are         optionally substituted with 1 to 3 substituents selected from         halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸.         —CO₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂;     -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,         C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,         C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the         C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,         C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R¹¹ and R¹²         are each optionally substituted with 1 to 3 substituents         independently selected from halogen, —CN, R⁸, —OR⁸, C(O)R⁸,         OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸, —NR⁸C(O)OR⁸,         —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸, —S(O)₂N(R⁹)₂,         —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and C₁-C₆haloalkoxy;     -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken         together with the N atom to which they are attached form an         optionally substituted C₃-C₈heterocycloalkyl ring optionally         containing an additional heteroatom selected from N, O and S;     -   ring A is an aryl or a heteroaryl, wherein the aryl and         heteroaryl groups of Ring A are optionally substituted with 1 to         3 R^(A) groups, wherein each R^(A) is independently selected         from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂, —CN,         —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂, —NR⁹C(S)N(R⁹)₂,         —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹)₂, —NR⁹NR⁹CO₂R⁸,         —C(O)C(O)R⁸, —C(O)CH2C(O)R⁸, —CO₂R⁸, —(CH₂)_(n)CO₂R⁸, —C(O)R⁸,         —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, —OC(O)N(R⁹)₂, —OC(O)R⁸,         —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)2R⁸, —S(O)₃R⁸, —SO₂N(R⁹)₂,         —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂,         —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸, —CH═CHCO₂R⁸,         —C(═NH)—N(R⁹)₂, and —(CH₂)_(n)NHC(O)R⁸ or two adjacent R^(A)         substituents on Ring A form a 5-6 membered ring that contains up         to two heteroatoms as ring members;     -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7         or 8;     -   each m is independently selected from 1, 2, 3, 4, 5 and 6, and     -   t is 1, 2, 3, 4, 5, 6, 7 or 8.         Formulae (C), (D), (E), (G) and (H)

As discussed above, the TLR agonist can be of formula (C), (D), (E), (G) or (H).

The ‘parent’ compounds of formulae (C), (D), (E) and (H) are useful TLR7 agonists (see references 7-10 and 32-48) but are preferably modified herein by attachment of a phosphorus-containing moiety.

In some embodiments of formulae (C), (D) and (E) the compounds have structures according to formulae (C′), (D′) and (E′), shown below:

The embodiments of the invention of formulae (C), (D), (E) and (H) also apply to formulae (C′), (D′), (E′) and (H′).

In some embodiments of formulae (C), (D), (E), and (H): X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl, CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and -Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C) is CH; X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; q is 1 or 2.

In other embodiments of formulae (C), (D), (E), and (H): X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl, CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and -Y-L-X—-P(O)(OR^(X))(OR^(Y)); X^(C) is N; X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.

In other embodiments of formula (D): P⁵ is selected from C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (D): X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (D): X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is O; L is selected from C₁-C₆alkylene and —((CH2)_(p)O)_(q)(CH2)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)2 and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X^(E) is CH₂, P⁸ is C₁-C₆alkoxy optionally substituted with -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (E): P⁹ is —NHC₁-C₆alkyl optionally substituted with OH and C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, a compound of formula (C) is not a compound in which P⁴ is -Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, in a compound of formula (C), P⁴ is selected from H, C₁-C₆alkyl, —C₁-C₆alkylaryl.

In some embodiments of formula (H): X^(H1)—X^(H2) is CR^(H2)R^(H3), R^(H2) and R^(H3) are H, X^(H3) is N, X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (H): X^(H1)—X^(H2) is CR^(H2)R^(H3), R^(H2) and R^(H3) are H, X^(H3) is N, X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)2 and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

The ‘parent’ compounds of formula (G) are useful TLR8 agonists (see references 11 & 12) but are preferably modified herein by attachment of a phosphorus-containing moiety to permit adsorption. In some embodiments of formula (G), the compounds have structures according to formula (G′);

In some embodiments of formula (G) or (G′): X^(G) is C and

represents a double bond.

In some embodiments of formula (G) or (G′): X is a covalent bond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (G) or (G′): X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2 and 3; and q is selected from 1 and 2.

Oil-in-Water Emulsion Adjuvants

According to the invention's second aspect a vaccine is adjuvanted with an oil-in-water emulsion. Various such emulsions are known e.g. MF59 and AS03 are both authorised in Europe.

Useful emulsion adjuvants they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion generally have a sub-micron diameter, and these small sizes can readily be achieved with a microfluidiser to provide stable emulsions, or by alternative methods e.g. phase inversion. Emulsions in which at least 80% (by number) of droplets have a diameter of less than 220 nm are preferred, as they can be subjected to filter sterilization.

The emulsion can include oil(s) from an animal (such as fish) and/or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolisable and may therefore be used with the invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.

Most fish contain metabolisable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred for use with the invention (see below). Squalane, the saturated analog to squalene, is also a useful oil. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.

Preferred amounts of total oil (% by volume) in an adjuvant emulsion are between 1 and 20% e.g. between 2-10%. A squalene content of 5% by volume is particularly useful.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10 e.g. about 15. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 or polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85) or sorbitan monolaurate.

Emulsions used with the invention preferably include non-ionic surfactant(s). Preferred surfactants for including in the emulsion are polysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80), Span 85 (sorbitan trioleate), lecithin or Triton X-100. Mixtures of surfactants can be used e.g. a mixture of polysorbate 80 and sorbitan trioleate. A combination of a polyoxyethylene sorbitan ester such as polysorbate 80 (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also useful . Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol. Where a mixture of surfactants is used then the HLB of the mixture is calculated according to their relative weightings (by volume) e.g. the preferred 1:1 mixture by volume of polysorbate 80 and sorbitan trioleate has a HLB of 8.4.

Preferred amounts of total surfactant (% by volume) in an adjuvant emulsion are between 0.1 and 2% e.g. between 0.25-2%. A total content of 1% by volume is particularly useful e.g. 0.5% by volume of polysorbate 80 and 0.5% by volume of sorbitan trioleate.

Useful emulsions can be prepared using known techniques e.g. see references 3 and 49-55.

Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:

-   -   A submicron emulsion of squalene, polysorbate 80, and sorbitan         trioleate. The composition of the emulsion by volume can be         about 5% squalene, about 0.5% polysorbate 80 and about 0.5%         sorbitan trioleate. In weight terms, these ratios become 4.3%         squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This         adjuvant is known as ‘MF59’ [54-56], as described in more detail         in Chapter 10 of ref. 2 and chapter 12 of ref. 3. The MF59         emulsion advantageously includes citrate ions e.g. 10 mM sodium         citrate buffer.     -   An emulsion of squalene, a tocopherol, and polysorbate 80. The         emulsion may include phosphate buffered saline. These emulsions         may have from 2 to 10% squalene, from 2 to 10% tocopherol and         from 0.3 to 3% polysorbate 80, and the weight ratio of         squalene:tocopherol is preferably ≤1 (e.g. 0.90) as this can         provide a more stable emulsion. Squalene and polysorbate 80 may         be present volume ratio of about 5:2, or at a weight ratio of         about 11:5. Thus the three components (squalene, tocopherol,         polysorbate 80) may be present at a weight ratio of         1068:1186:485 or around 55:61:25. This adjuvant is known as         ‘AS03’. Another useful emulsion of this type may comprise, per         human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4         mg polysorbate 80 [57] e.g. in the ratios discussed above.     -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol         (e.g. a cholesterol) are associated as helical micelles [58].     -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a         phospholipid, and 0.05-5% of a non-ionic surfactant. As         described in reference 59, preferred phospholipid components are         phosphatidylcholine, phosphatidylethanolamine,         phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,         phosphatidic acid, sphingomyelin and cardiolipin. Submicron         droplet sizes are advantageous.     -   An emulsion comprising squalene, an aqueous solvent, a         polyoxyethylene alkyl ether hydrophilic nonionic surfactant         (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic         nonionic surfactant (e.g. a sorbitan ester or mannide ester,         such as sorbitan monoleate or ‘Span 80’). The emulsion is         preferably thermoreversible and/or has at least 90% of the oil         droplets (by volume) with a size less than 200 nm [60]. The         emulsion may also include one or more of: alditol; a         cryoprotective agent (e.g. a sugar, such as dodecylmaltoside         and/or sucrose); and/or an alkylpolyglycoside. It may also         include a TLR4 agonist, such as one whose chemical structure         does not include a sugar ring [61]. Such emulsions may be         lyophilized. The ‘AF03’ product is one such emulsion.

Preferred oil-in-water emulsions used with the invention comprise squalene and polysorbate 80.

The emulsions may be mixed with TdaP antigens during vaccine manufacture, or they may be mixed extemporaneously at the time of delivery. Thus, in some embodiments, the adjuvant and antigens may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. At the time of mixing (whether during bulk manufacture, or at the point of use) the antigen will generally be in an aqueous form, such that the final vaccine is prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1. If emulsion and antigen are stored separately in a kit then the product may be presented as a vial containing emulsion and a vial containing aqueous antigen, for mixing to give adjuvanted liquid vaccine (monodose or multi-dose).

Preferred emulsions of the invention include squalene oil. This is usually prepared from shark oil but alternative sources are known e.g. see references 62 (yeast) and 63 (olive oil). Squalene which contains less than 661 picograms of PCBs per gram of squalene (TEQ) is preferred for use with the invention, as disclosed in reference 64. The emulsions are preferably made from squalene of high purity e.g. prepared by double-distillation as disclosed in reference 65.

Where a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols are preferred. The tocopherol can take several forms e.g. different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-α-tocopherol and DL-α-tocopherol can both be used. Tocopherols have antioxidant properties that may help to stabilize the emulsions [66]. A preferred α-tocopherol is DL-α-tocopherol, and a preferred salt of this tocopherol is the succinate.

Diphtheria Toxoid

Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive non-sporing aerobic bacterium. This organism expresses a prophage-encoded ADP-ribosylating exotoxin (‘diphtheria toxin’), which can be treated (e.g. using formaldehyde) to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. Diphtheria toxoids are disclosed in more detail in chapter 13 of reference 67. Preferred diphtheria toxoids are those prepared by formaldehyde treatment. The diphtheria toxoid can be obtained by growing C. diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation. The toxoided material may then be treated by a process comprising sterile filtration and/or dialysis.

Quantities of diphtheria toxoid can be expressed in international units (IU). For example, the NIBSC [68] supplies the ‘Diphtheria Toxoid Adsorbed Third International Standard 1999’ [69,70], which contains 160 IU per ampoule. As an alternative to the IU system, the ‘Lf’ unit (“flocculating units”, the “limes flocculating dose”, or the “limit of flocculation”) is defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture [71]. For example, the NIBSC supplies ‘Diphtheria Toxoid, Plain’ [72], which contains 300 Lf per ampoule and ‘The 1st International Reference Reagent For Diphtheria Toxoid For Flocculation Test’ [73] which contains 900 Lf per ampoule. The concentration of diphtheria toxin in a composition can readily be determined using a flocculation assay by comparison with a reference material calibrated against such reference reagents. The conversion between IU and Lf systems depends on the particular toxoid preparation.

The concentration of diphtheria toxoid in a composition of the invention is typically in the range of 2-8 Lf/ml (e.g. 4 Lf/ml or 5 Lf/ml), but is ideally ≤4 Lf/ml. In a typical 0.5 ml unit dose volume, therefore, the amount of diphtheria toxoid can be 2 Lf, 2.5 Lf, or less than 2 Lf.

Diphtheria toxoid in the composition is preferably adsorbed (more preferably totally adsorbed) onto an aluminium salt, preferably onto an aluminium hydroxide adjuvant.

Tetanus Toxoid

Tetanus is caused by Clostridium tetani, a Gram-positive, spore-forming bacillus. This organism expresses an endopeptidase (‘tetanus toxin’), which can be treated to give a toxoid that is no longer toxic but that remains antigenic and is able to stimulate the production of specific anti-toxin antibodies after injection. Tetanus toxoids are disclosed in more detail in chapter 27 of reference 67. Preferred tetanus toxoids are those prepared by formaldehyde treatment. The tetanus toxoid can be obtained by growing C. tetani in growth medium (e.g. a Latham medium derived from bovine casein), followed by formaldehyde treatment, ultrafiltration and precipitation. The material may then be treated by a process comprising sterile filtration and/or dialysis.

Quantities of tetanus toxoid can be expressed in international units (IU). For example, NIBSC supplies the ‘Tetanus Toxoid Adsorbed Third International Standard 2000’ [74,75], which contains 469 IU per ampoule. As with diphtheria toxoid, the ‘Lf’ unit is an alternative to the IU system. NIBSC supplies ‘The 1st International Reference Reagent for Tetanus Toxoid For Flocculation Test’ [76] which contains 1000 LF per ampoule. The concentration of diphtheria toxin in a composition can readily be determined using a flocculation assay by comparison with a reference material calibrated against such reference reagents.

The concentration of tetanus toxoid in a composition of the invention is typically in the range of 5-15 Lf/ml (e.g. 10 Lf/ml), but is ideally ≤9 Lf/ml. In a typical 0.5 ml unit dose volume, therefore, the amount of diphtheria toxoid can be 5 Lf, or less than 4.5 Lf.

Tetanus toxoid in the composition is preferably adsorbed (sometimes totally adsorbed) onto that salt, preferably onto an aluminium hydroxide adjuvant.

Pertussis Toxoid

Bordetella pertussis causes whooping cough. Pertussis antigens in vaccines are either cellular (whole cell, in the form of inactivated B. pertussis cells; ‘wP’) or acellular (‘aP’). Preparation of cellular pertussis antigens is well documented (e.g. see chapter 21 of reference 67) e.g. it may be obtained by heat inactivation of phase I culture of B. pertussis. The invention preferably uses acellular antigens, including, one, two or (preferably) three of the following three antigens: (1) detoxified pertussis toxin (pertussis toxoid, or ‘PT’); (2) filamentous hemagglutinin (‘FHA’); (3) pertactin (also known as the ‘69 kiloDalton outer membrane protein’). These three antigens can be prepared by isolation from B. pertussis culture grown in modified Stainer-Scholte liquid medium. PT and FHA can be isolated from the fermentation broth (e.g. by adsorption on hydroxyapatite gel), whereas pertactin can be extracted from the cells by heat treatment and flocculation (e.g. using barium chloride). The antigens can be purified in successive chromatographic and/or precipitation steps. PT and FHA can be purified by hydrophobic chromatography, affinity chromatography and size exclusion chromatography. Pertactin can be purified by ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography, or by IMAC. FHA and pertactin may be treated with formaldehyde prior to use according to the invention. PT is preferably detoxified by treatment with formaldehyde and/or glutaraldehyde. As an alternative to this chemical detoxification procedure the PT may be a mutant PT in which enzymatic activity has been reduced by mutagenesis [77] (e.g. the 9K/129G double mutant [78]).

The invention preferably uses a PT-containing aP antigen. When using an aP antigen a composition of the invention will typically, in addition to the PT, include FHA and, optionally, pertactin. It can also optionally include fimbriae types 2 and 3.

Quantities of acellular pertussis antigens are typically expressed in micrograms. The concentration of pertussis toxoid in a composition of the invention is typically in the range of 2-20 μg/ml (e.g. 5 μg/ml or 16 μg/ml), but is ideally ≤4 μg/ml. In a typical 0.5 ml unit dose volume, therefore, the amount of diphtheria toxoid can be 2.5 μg, 8 μg, or <2 μg.

It is usual that each of pertussis toxoid, FHA and pertactin are present in a composition of the invention. These may be present at various ratios (by mass), such as PT:FHA:p69 ratios of 16:16:5 or 5:10:6. Each of these three antigens will generally be present at <20 μg/ml e.g. each in the range of 4-20 μg/ml. A total pertussis antigen concentration of <40 μg/ml is typical. It is usual to have a mass excess of FHA relative to pertactin.

Pertussis toxoid in the composition is preferably adsorbed (sometimes totally adsorbed) onto that salt, preferably onto an aluminium hydroxide adjuvant. Any FHA can also be adsorbed to an aluminium hydroxide adjuvant. Any pertactin can be adsorbed to an aluminium phosphate adjuvant.

Inactivated Poliovirus Antigen (IPV)

Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. As explained in chapter 24 of reference 67, it is therefore preferred to use three poliovirus antigens with the invention—poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain). As an alternative to these strains (“Salk” strains), Sabin strains of types 1 to 3 can be used e.g. as discussed in references 79 & 80. These strains can be more potent than the normal Salk strains.

Polioviruses may be grown in cell culture. A preferred culture uses a Vero cell line, which is a continuous cell line derived from monkey kidney. Vero cells can conveniently be cultured microcarriers. Culture of the Vero cells before and during viral infection may involve the use of bovine-derived material, such as calf serum, and of lactalbumin hydrolysate (e.g. obtained by enzymatic degradation of lactalbumin). Such bovine-derived material should be obtained from sources which are free from BSE or other TSEs. The final vaccine preferably contains less than 10 ng/ml, preferably ≤1 ng/ml e.g. ≤500 pg/ml or ≤50 pg/ml of Vero cell DNA e.g. less than 10 ng/ml of Vero cell DNA that is ≥50 base pairs long.

After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Prior to administration to patients, polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde before the viruses are used in the process of the invention.

The viruses are preferably grown, purified and inactivated individually, and are then combined to give a bulk mixture for use with the invention.

Quantities of inactivated poliovirus (IPV) are typically expressed in the ‘DU’ unit (the “D-antigen unit” [81]). Typically a composition has IPV antigens 1/2/3 at concentrations of 80/16/64 DU/ml (40/8/32 DU per 0.5 ml dose). In some embodiments, however, a composition can include lower amounts of poliovirus antigens, either through including less antigen or by using more potent strains. For a Type 1 poliovirus the concentration of the virus in the composition can be ≤20 DU/ml e.g. <18, <16, <14, <12, <10, etc. For a Type 2 poliovirus the concentration of the virus in the composition can be ≤4 DU/ml e.g. <3, <2, <1, <0.5, etc. For a Type 3 poliovirus the concentration of the virus in the composition can be ≤16 DU/ml e.g. <14, <12, <10, <8, <6, etc. Where all three of Types 1, 2 and 3 poliovirus are present the three antigens can be present at a DU ratio of 5:1:4 respectively, or at any other suitable ratio e.g. a ratio of 15:32:45 when using Sabin strains [79]. A low dose of antigen from Sabin strains is particularly useful, with ≤10 DU type 1, ≤20 DU type 2, and ≤30 DU type 3 (per unit dose).

Polioviruses are preferably not adsorbed to any adjuvant before they are formulated, but after formulation they may become adsorbed onto aluminium salt(s) in the composition.

Combination Vaccines

As well as including D, T, Pa, and/or poliovirus antigens, immunogenic compositions of the invention may include antigens from further pathogens. For example, these antigens may be HBsAg, conjugated Hib capsular saccharide, conjugated N.meningitidis capsular saccharide (one or more of serogroups A, C, W135 and/or Y) or conjugated S.pnetimonide capsular saccharide. For example, any of the suitable antigen components of PEDIARIX, MENVEO, MENACTRA, NIMENRIX, PREVNAR, or SYNFLORIX can be used.

Preferably, however, the vaccine's only antigenic components are either (i) for diphtheria, tetanus and pertussis or (ii) for diphtheria, tetanus, pertussis and poliovirus.

Immunogenic compositions of the invention include at least a diphtheria toxoid, a tetanus toxoid, and a pertussis toxoid as immunogenic components. The compositions include an excess of tetanus toxoid relative to diphtheria toxoid. This excess is measured in Lf units (but the excess can also be seen via IU e.g. Adacel's content is quoted as 5 Lf of tetanus toxoid and 2 Lf of diphtheria toxoid, or as 20IU of tetanus toxoid and 2 IU of diphtheria toxoid). The excess is ideally at least 1.5:1 (i.e. at least 1.5 Lf of tetanus toxoid for every 1 Lf of diphtheria toxoid) e.g. 2:1 or 2.5:1. The excess will not usually be more than 5-fold (again, in Lf units).

As an independent embodiment of the disclosure, the invention provides an immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid (e.g. the PT-9K/129G double mutant), filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2. For instance, the composition can comprise 4 μg pertussis toxoid, 4 μg FHA and 8 μg pertactin per unit dose. The composition can also include diphtheria toxoid (e.g. 2 Lf per unit dose) and tetanus toxoid (e.g. 5 Lf per dose) to provide a TdaP combination. The composition can also include an adjuvant comprising an aluminium salt (e.g. an aluminium hydroxide) and, optionally, a TLR agonist as described elsewhere herein.

Pharmaceutical Compositions and Products

The invention provides various immunogenic compositions. These are ideally pharmaceutical compositions suitable for use in humans. Pharmaceutical compositions usually include components in addition to the immunogen and the adjuvant components e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference 82.

Pharmaceutical compositions are preferably in aqueous form, particularly at the point of administration, but they can also be presented in non-aqueous liquid forms or in dried forms e.g. as gelatin capsules, or as lyophilisates, etc.

Pharmaceutical compositions may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. Pharmaceutical compositions can include a physiological salt, such as a sodium salt e.g. to control tonicity. Sodium chloride (NaCl) is typical, which may be present at between 1 and 20 mg/ml e.g. 10±2 mg/ml or 9 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.

Pharmaceutical compositions can have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg. Compositions may be isotonic with humans.

Pharmaceutical compositions may include compounds (with or without an insoluble aluminium salt) in plain water (e.g. w.f.i.) but will usually include one or more buffers. Typical buffers include: a phosphate buffer (except in the fifteenth aspect); a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminium hydroxide adjuvant); or a citrate buffer. Buffer salt s will typically be included in the 5-20mM range. If a phosphate buffer is used then the concentration of phosphate ions should, in some embodiments, be <50 mM (see above) e.g. <10 mM.

The pH of a composition of the invention will generally be between 6.0 and 7.5. A manufacturing process may therefore include a step of adjusting the pH of a composition prior to packaging. Aqueous compositions administered to a patient can have a pH of between 5.0 and 7.5, and more typically between 5.0 and 6.0 for optimum stability; where a diphtheria toxoid and/or tetanus toxoid is present, the pH is ideally between 6.0 and 7.0.

Pharmaceutical compositions are preferably sterile.

Pharmaceutical compositions preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. 1 EU is equal to 0.2 ng FDA reference standard Endotoxin EC-2 ‘RSE’) per dose.

Pharmaceutical compositions are preferably gluten free.

During manufacture, dilution of components to give desired final concentrations will usually be performed with WFI (water for injection).

Pharmaceutical compositions are suitable for administration to animal (and, in particular, human) patients, and thus include both human and veterinary uses. They may be used in a method of raising an immune response in a patient, comprising the step of administering the composition to the patient.

The invention can provide bulk material which is suitable for packaging into individual doses, which can then be distributed for administration to patients. Concentrations discussed above are typically concentrations in final packaged dose, and so concentrations in bulk vaccine may be higher (e.g. to be reduced to final concentrations by dilution).

Pharmaceutical compositions may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.05-1.5 ml e.g. about 0.5 ml for intramuscular injection. References to 0.5 ml doses will be understood to include normal variance e.g. 0.5 ml±0.05 ml. For multidose situations, multiple dose amounts will be extracted and packaged together in a single container e.g. 5 ml for a 10-dose multidose container (or 5.5 ml with 10% overfill).

The invention also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) containing a pharmaceutical composition of the invention e.g. containing a unit dose. This device can be used to administer the composition to a vertebrate subject.

The invention also provides a sterile container (e.g. a vial) containing a pharmaceutical composition of the invention e.g. containing a unit dose.

The invention also provides a unit dose of a pharmaceutical composition of the invention.

The invention also provides a hermetically sealed container containing a pharmaceutical composition of the invention. Suitable containers include e.g. a vial.

Pharmaceutical compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition), although aqueous compositions are preferred. Suspensions for intramuscular or intradermal or subcutaneous injection are typical.

Compositions of the first aspect comprise an effective amount of a TLR agonist i.e. an amount which, when administered to an individual, either in a single dose or as part of a series, is effective for enhancing the immune response to a its co-administered immunogens. This amount can vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors.

The amount of TLR agonist in a unit dose of a composition will fall in a relatively broad range that can be determined through routine trials. An amount of between 1-1000 μg/dose can be used e.g. from 5-100 μg per dose or from 10-100 μg per dose, and ideally ≤300 μg per dose e.g. about 5 μg, 10 μg, 20 μg, 25 μg, 50 μg or 100 μg per dose. Thus the concentration of a TLR agonist in a composition of the invention may be from 2-2000 μg/ml e.g. from 10-200 μg/ml, or about 5, 10, 20, 40, 50, 100 or 200 μg/ml, and ideally ≤600 μg/ml.

Methods of Treatment, and Administration of Immunogenic Compositions

The invention is suitable for raising immune responses in humans, but they may also be useful in non-human animals (in particular mammals) subjects. Compositions prepared according to the invention may be used to treat both children and adults.

The invention provides a method of raising an immune response in a subject, comprising the step of administering to the subject a composition of the invention. The invention also provides a composition of the invention, for use in a method of raising an immune response in a subject. The invention also provides the use of (i) a TLR agonist as defined herein and (ii) an insoluble aluminium salt and (iii) at least diphtheria, tetanus and pertussis toxoids, in the manufacture of a medicament (e.g. a vaccine) for raising an immune response in a subject. The invention also provides the use of (i) an oil-in-water emulsion adjuvant as defined herein and (ii) at least diphtheria, tetanus and pertussis toxoids, in the manufacture of a medicament (e.g. a vaccine) for raising an immune response in a subject.

The immune response stimulated by these methods and uses will generally include an antibody response, preferably a protective antibody response. The immune response can also include a cellular response. Methods for assessing antibody and cellular immune responses after immunisation are well known in the art, particularly for diphtheria, tetanus, pertussis and poliovirus antigens.

Administration of compositions of the invention will generally be by injection, and this may be by the subcutaneous, intradermal of the intramuscular route. Intramuscular injection is preferred.

Immunogenic compositions of the invention will generally be administered to people at least 3 years of age, and preferably at least 4 years of age. For instance, the subject may be 10-64 years old, or a teenager. The compositions are most useful in people who have previously received routine childhood immunisations (including DTP vaccine).

A patient will generally receive the composition as frequently as required. Each course of immunisation will involve a single booster dose.

Chemical Groups

Unless specifically defined elsewhere, the chemical groups discussed herein have the following meaning when used in present specification:

The term “alkyl” includes saturated hydrocarbon residues including:

-   -   linear groups up to 10 atoms (C₁-C₁₀), or of up to 6 atoms         (C₁-C₆), or of up to 4 atoms (C₁-C₄). Examples of such alkyl         groups include, but are not limited, to C₁-methyl, C₂-ethyl,         C₃-propyl and C₄-n-butyl.     -   branched groups of between 3 and 10 atoms (C₃-C₁₀), or of up to         7 atoms (C₃-C₇), or of up to 4 atoms (C₃-C₄). Examples of such         alkyl groups include, but are not limited to, C₃-iso-propyl,         C₄-sec-butyl, C₄-iso-butyl, C₄-tert-butyl and C₅-neo-pentyl.

The term “alkylene” refers to the divalent hydrocarbon radical derived from an alkyl group, and shall be construed in accordance with the definition above.

The term “alkenyl” includes monounsaturated hydrocarbon residues including:

-   -   linear groups of between 2 and 6 atoms (C2-C₆). Examples of such         alkenyl groups include, but are not limited to, C₂-vinyl,         C₃-1-propenyl, C₃-allyl, C₄-2-butenyl     -   branched groups of between 3 and 8 atoms (C₃-C₈). Examples of         such alkenyl groups include, but are not limited to,         C₄-2-methyl-2-propenyl and C₆-2,3-dimethyl-2-butenyl.

The term alkenylene refers to the divalent hydrocarbon radical derived from an alkenyl group, and shall be construed in accordance with the definition above.

The term “alkoxy” includes 0-linked hydrocarbon residues including:

-   -   linear groups of between 1 and 6 atoms (C₁-C₆), or of between 1         and 4 atoms (C₁-C₄). Examples of such alkoxy groups include, but         are not limited to, C₁-methoxy, C₂-ethoxy, C₃-n-propoxy and         C₄-n-butoxy.     -   branched groups of between 3 and 6 atoms (C₃-C₆) or of between 3         and 4 atoms (C₃-C₄).

Examples of such alkoxy groups include, but are not limited to, C₃-iso-propoxy, and C₄-sec-butoxy and tert-butoxy.

Halo is selected from Cl, F, Br and I. Halo is preferably F.

The term “aryl” includes a single or fused aromatic ring system containing 6 or 10 carbon atoms; wherein, unless otherwise stated, each occurrence of aryl may be optionally substituted with up to 5 substituents independently selected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, halo, CN, COOR¹⁴, CF₃ and NR¹⁴R¹⁵; as defined above. Typically, aryl will be optionally substituted with 1, 2 or 3 substituents. Optional substituents are selected from those stated above. Examples of suitable aryl groups include phenyl and naphthyl (each optionally substituted as stated above). Arylene refers the divalent radical derived from an aryl group, and shall be construed in accordance with the definition above.

The term “heteroaryl” includes a 5, 6, 9 or 10 membered mono- or bi-cyclic aromatic ring, containing 1 or 2 N atoms and, optionally, an NR¹⁴ atom, or one NR¹⁴ atom and an S or an O atom, or one S atom, or one O atom; wherein, unless otherwise stated, said heteroaryl may be optionally substituted with 1, 2 or 3 substituents independently selected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, halo, CN, COOR¹⁴, CF₃ and NR¹⁴R¹⁵; as defined below. Examples of suitable heteroaryl groups include thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, benzotriazolyl, quinolinyl and isoquinolinyl (optionally substituted as stated above).

Heteroarylene refers the divalent radical derived from heteroaryl, and shall be construed in accordance with the definition above.

The term “heterocyclyl” is a C-linked or N-linked 3 to 10 membered non-aromatic, mono- or bi-cyclic ring, wherein said heterocycloalkyl ring contains, where possible, 1, 2 or 3 heteroatoms independently selected from N, NR¹⁴, S(O)_(q) and O; and said heterocycloalkyl ring optionally contains, where possible, 1 or 2 double bonds, and is optionally substituted on carbon with 1 or 2 substituents independently selected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, CN, CF₃, halo, COOR¹⁴, NR¹⁴R¹⁵ and aryl.

In the above definitions R¹⁴ and R¹⁵ are independently selected from H and (C₁-C₆)alkyl.

When a structural formula is defined with a substituent attached to the core of the molecule by an unspecified, or “floating” bond, for example, as for the group P³ in the case of formula (C), this definition encompasses the cases where the unspecified substituent is attached to any of the atoms on the ring in which the floating bond is located, whilst complying with the allowable valence for that atom.

In the case of compounds of the invention which may exist in tautomeric forms (i.e. in keto or enol forms), for example the compounds of formula (C) or (H), reference to a particular compound optionally includes all such tautomeric forms.

General

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X +Y.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

The term “about” in relation to a numerical value x is optional and means, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

As animal (and particularly bovine) materials are typically used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE).

Where a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.

Phosphorous-containing groups employed with the invention may exist in a number of protonated and deprotonated forms depending on the pH of the surrounding environment, for example the pH of the solvent in which they are dissolved. Therefore, although a particular form may be illustrated it is intended, unless otherwise mentioned, for these illustrations to merely be representative and not limiting to a specific protonated or deprotonated form. For example, in the case of a phosphate group, this has been illustrated as —OP(O)(OH)₂ but the definition includes the protonated forms —[OP(O)(OH₂)(OH)]⁺ and —[OP(O)(OH₂)₂]²⁺ that may exist in acidic conditions and the deprotonated forms —[OP(O)(OH)(O)]⁻ and [OP(O)(O)₂]²⁻ that may exist in basic conditions.

Compounds disclosed herein can exist as pharmaceutically acceptable salts. Thus, the compounds may be used in the form of their pharmaceutically acceptable salts i.e. physiologically or toxicologically tolerable salt (which includes, when appropriate, pharmaceutically acceptable base addition salts and pharmaceutically acceptable acid addition salts).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the % of FHA-specific memory B cells for the indicated treatment groups.

MODES FOR CARRYING OUT THE INVENTION

Vaccine Preparation

References 31 and 83 disclose TLR7 agonists having formula (K) as discussed above. One of these compounds, 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo [f]-[1,7]naphthyridin-8-yl)propanoic acid is referred to hereafter as compound “K2”:

Compound K2 is added to water at 4mg/ml, then 1M NaOH is added to ensure full solubilisation, with stirring for 15 minutes at room temperature. This material is added to a suspension of aluminium hydroxide adjuvant (Al—H) to give the desired final concentration. This mixture is shaken for 2 hours at ambient temperature to ensure full adsorption, and then histidine buffer components are added (10mM histidine buffer, pH 6.5).

The compound can also be used as an arginine salt monohydrate (obtained by mixing 98 mg of the compound with 1.7 ml of 0.1 M arginine in 80/20 methanol/water to give a 57 mg/ml solution, followed by addition of 7 ml ethanol to precipitate the salt) in which case it is seen that the NaOH is not required for solubilisation prior to mixing with the Al—H.

Four different mixtures are prepared, giving a final K2 concentration of 10, 50, 250 or 500 μg/ml (to provide a 1, 5, 25 or 50 μg dose of K2 in a 100 μl dosage volume); the Al—H concentration is always 3 mg/ml. At all strengths >95% of compound K2 is adsorbed to the Al—H. The adsorbed adjuvant is referred to hereafter as “Al—H/K2”.

Adjuvant Adsorption to Antigens

3-valent (DTaP) vaccines were adjuvanted with Al—H alone or with Al—H/K2. These formulations showed optimal pH (6.5-6.8±0.1) and osmolarity values (0.300±50 mO). Osmolarity was adjusted with NaCl. Adsorption was detected by separating the adjuvant-antigen complexes from unadsorbed antigens by centrifugation. 0.4% DOC was added to the supernatant containing the unadsorbed antigens. Antigens were precipitated by the addition of 60% TCA and collected by centrifugation. The pellet containing the TCA-precipitated antigens was resuspended in loading buffer and loaded onto an SDS-PAGE gel. The pellet containing the adjuvant-antigen complexes was resuspended in desorption buffer (4× concentration: 0.5 M Na2HPO₄ pH, 8 g SDS, 25 g glycerol, 6.16 g DTT and bromophenol blue), the aluminium hydroxide was removed by centrifugation and the supernatant applied to an SDS-PAGE gel.

Using Al—H alone at a concentration of 2 mg/ml, the adsorption profiles for DT, TT, PT, FHA and 69K detected by Coomassie Blue staining were complete. No bands were detected in the DOC-TCA-treated supernatants. Western Blot analysis confirmed complete Al—H adsorption for DT, TT, PT, FHA and 69K.

Four different K2 concentrations were tested (0.1, 0.025, 0.01, 0.005 mg/ml). The Al—H concentration was constant at 2 mg/ml. Even at 0.1 mg/ml K2 all antigens were completely adsorbed.

Immunogenicity Testing with TLR Agonists

Four vaccines were tested, each containing (per 0.5 ml) 5 Lf tetanus toxoid, 2 Lf of diphtheria toxoid, and 16 μg acellular pertussis antigens (a mixture of purified PT-9K/129G, FHA and p69 pertactin).

The four vaccines were (A) unadjuvanted (B) adjuvanted with 2 mg/ml Al—H (C) adjuvanted with 2 mg/ml Al—H plus 100 μg/ml synthetic monophosphoryl lipid A i.e. a TLR4 agonist, or (D) adjuvanted with 2 mg/ml Al—H plus 1 mg/ml compound ‘K2’ i.e. a TLR7 agonist. The TLR agonists in vaccines (C) and (D) were adsorbed to the Al—H. All antigens were adsorbed to the Al—H in formulations (B), (C) and (D).

For comparison the BOOSTRIX™ product was also tested. As discussed above it contains (per 0.5 ml) 2.5 Lf of diphtheria toxoid, 5 Lf tetanus toxoid, and 18.5 μg acellular pertussis antigens (a mixture of purified PT, FHA and p69 pertactin), and it is adjuvanted with a mixture of aluminium phosphate and hydroxide salts. A mixture of buffer and Al—H was used as a negative control.

The four vaccines were administered to female Balb/C mice (6 weeks old) at 100 μl intramuscular doses on days 0, 21 and 35. Sera were tested 2 weeks after each dose.

Serum total IgG titers were measured against each antigen and were as follows (geometric means):

Day Ag Unadj Al—H Al—H + K2 Al—H + MPL Boostrix −ve control 14 Dt 0.030 0.603 8.119 1.762 1.205 0.030 Tt 0.191 2.546 46.14 14.49 3.217 0.030 PT 14.28 10.26 26.68 16.61 4.685 1.080 FHA 0.145 0.579 27.38 7.942 1.458 0.126 p69 0.566 7.046 72.47 38.49 12.39 0.129 35 Dt 0.030 69.29 490.1 139.3 89.59 0.030 Tt 48.98 128.6 808.9 298.0 109.8 0.030 PT 245.0 267.5 377.1 695.9 195.0 2.734 FHA 7.847 65.25 400.6 231.0 49.49 0.030 p69 32.50 222.2 1484 575.8 318.0 0.050 49 Dt 0.055 79.74 452.9 149.2 88.95 0.047 Tt 60.59 123.2 694.3 317.0 105.6 0.030 PT 200.8 387.7 329.7 642.3 300.0 2.065 FHA 20.96 72.10 462.4 272.5 78.98 0.030 p69 107.6 384.9 1275 794.5 302.6 0.134

Thus in all cases and at all time points (except for PT at day 49) the highest titers in these 6 groups were seen in the mice which had received the antigens adjuvanted with adsorbed TLR agonist, and the addition of a TLR agonist to Al—H improved IgG responses relative to Al—H alone. Importantly, improved responses were seen in all cases when compared to the licensed BOOSTRIX™ vaccine. Moreover, unlike Al—H alone or BOOSTRIX™, the adsorbed TLR agonists were consistently able to improve anti-PT titers relative to the unadjuvanted group.

The use of the TLR agonists also leads to more rapid responses. The second dose showed a clear increase in IgG responses for all antigens, but the improvements after the third dose were not so significant. The mice in these experiments were naive to DTP but in a real-world human situation the target patients will previously have received DTP vaccine as a child and so the rapid response seen after the second dose in these experiments is helpful.

Immunogenicity Testing with Oil-in-Water Emulsion

Vaccines were prepared containing (per 0.5 ml) 5 Lf tetanus toxoid, 2 Lf of diphtheria toxoid, and 16 μg acellular pertussis antigens (a mixture of purified PT-9K/129G, FHA and p69 pertactin). These were administered to female Balb/C mice (6 weeks old) at 100 μl intramuscular doses on days 0, 21 and 35. Sera were tested 2 weeks after each dose. Vaccines were (A) unadjuvanted or (B) adjuvanted with MF59 emulsion, by mixing 50 μl antigen solution with 50 μl MF59. For comparison the BOOSTRIX™ product was also tested, and an antigen-free negative control was also tested.

Serum total IgG titers were measured against each antigen and were as follows (geometric means):

Day Ag Unadj MF59 Boostrix −ve control 14 Dt 0.030 0.038 1.308 0.030 Tt 0.064 3.025 2.480 0.030 PT 7.255 9.448 1.629 1.517 FHA 0.034 0.026 0.844 0.137 p69 1.365 9.281 14.46 1.670 35 Dt 0.030 115.2 85.55 0.033 Tt 24.31 379.6 83.61 0.060 PT 247.4 391.9 112.7 3.582 FHA 7.433 148.8 37.58 0.068 p69 11.44 1006 398.3 3.817 49 Dt 0.5165 78.54 92.18 0.054 Tt 40.21 337.5 99.09 0.030 PT 353.6 480.8 162.8 5.231 FHA 13.32 218.1 65.34 0.119 p69 36.91 1036 403.6 1.952

Thus after two doses in DTP-naive mice the emulsion-adjuvanted vaccine gave much better antibody titers than the approved BOOSTRIX™ product. This superiority was maintained after a third dose, except for the anti-Dt response. Moreover, unlike BOOSTRIX™, the emulsion was able to improve anti-PT titers relative to the unadjuvanted group.

FHA-Specific Memory B Cells

Four-to-five months after the third dose, FHA-specific memory B cells were measured in the immunised mice. The mice were sacrificed and their spleen cells were cultured in the presence of IL-2 and CpG for 5 days in order to expand all memory B cells. Spleen cells were then harvested and seeded in 96-well ELISPOT plates previously coated with either FHA antigen (10 mg/ml) or anti-mouse Ig. After overnight incubation, plates were washed to remove unattached spleen cells and both FHA-specific and total memory B cells were detected by biotinylated anti-mouse Ig and HRP-streptavidin. Colored spots, representing individual memory B cell, were counted with an ELISPOT reader instrument. The percentage of FHA-specific B cells compared to total B cells was then calculated for each sample, and FIG. 1 shows the results for the following groups: (A) unadjuvanted; (B) adjuvanted with Al—H; (C) adjuvanted with Al—H plus MPL-A; (D) adjuvanted with Al—H plus ‘K2’; (E) adjuvanted with MF59; (F) BOOSTRIX™; and (G) negative control. The highest response was seen in group (D) i.e. using the adsorbed TLR7 agonist. The next highest responses were seen in groups (C) and (E), with either the adsorbed TLR4 agonist or the emulsion, where responses were essentially the same but were still higher than with the BOOSTRIX™ product.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

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The invention claimed is:
 1. An immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, an aluminium salt adjuvant, and a Toll-like receptor (TLR) agonist, wherein the immunogenic composition comprises an excess as measured in limit of flocculation (Lf) units of tetanus toxoid relative to diphtheria toxoid.
 2. The immunogenic composition of claim 1, wherein the TLR agonist is a TLR4 agonist or a TLR 7 agonist.
 3. The immunogenic composition of claim 1, wherein the TLR agonist is adsorbed to the aluminium salt adjuvant.
 4. The immunogenic composition of claim 1, wherein 1, 2 or 3 of the toxoids is/are adsorbed to the aluminium salt adjuvant.
 5. The immunogenic composition of claim 1, with an Al+++ concentration ≤0.5 mg/ml.
 6. The immunogenic composition of claim 1, wherein the aluminium salt is an aluminium hydroxide.
 7. The immunogenic composition of claim 1, wherein the TLR agonist is a compound of formula (K), or a pharmaceutically acceptable salt thereof, Formula (K):

wherein: R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, —L¹R⁵, —L¹R⁶, —L²R⁵, —L²R⁶, —OL²R⁵, or —OL²R⁶; L¹ is —C(O)—or —O—; L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p), wherein the C₁-C₆alkylene and C₂-C₆alkenylene of L² are optionally substituted with 1 to 4 fluoro groups; each L³ is independently selected from C₁-C₆alkylene and —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³ is optionally substituted with 1 to 4 fluoro groups; L⁴ is arylene or heteroarylene; R² is H or C₁-C₆alkyl; R³ is selected from C₁-C₄alkyl, —L³R⁵, —L¹R⁵, —L³R⁷, —L³L⁴L³R⁷, —L³L⁴R⁵, —L³L⁴L³R⁵, —OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR⁸, —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH; each R⁴ is independently selected from H and fluoro; R⁵ is —P(O)(OR⁹)₂, R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰; R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰; R⁸ is H or C₁C₄alkyl; each R⁹ is independently selected from H and C₁-C₆alkyl; R¹⁰ is H or C₁-C₄alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or
 4. 8. The immunogenic composition of claim 1, wherein the TLR agonist is compound K2,


9. The immunogenic composition of claim 1, wherein the TLR agonist comprises at least one adsorptive moiety which allows it to adsorb to insoluble metal salts.
 10. The immunogenic composition of claim 9, wherein the adsorptive moiety is a phosphate or a phosphonate.
 11. The immunogenic composition of claim 1, wherein the TLR agonist has formula (C), (D), (E), (F), (G), (H), (I), (II), (J) or (K) as defined below, or is a 3-0-deacylated monophosphoryl lipid A (3d-MPL), Formulae (C), (D), (E), and (H) are as follows:

wherein: P³ is selected from H, C₁-C₆alkyl, CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and -Y-L-X —P(O)(OR^(X))(OR^(Y)); and P⁴ is selected from H, C₁-C₆alkyl, —C₁-C₆alkylaryl and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P³ and P⁴ is -Y-L-X—P(O)(OR^(X))(OR^(Y)), ⁵ is selected from H, C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(x))(OR^(Y)); P⁶ is selected from H, C₁-C₆alkyl each optionally substituted with 1 to 3 substituents selected from C₁-C₄alkyl and OH, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷ is selected from H, C₁-C₆alkyl, —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)—, —NHC₁-C₆alkyl and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P⁵, P⁶ and P⁷ is -Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁸ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, —NHC₁-C₆alkyl each optionally substituted with OH, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are each independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, —NHC₁-C₆alkyl each optionally substituted with OH and C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P^(8,) P⁹ or P¹⁰ is -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁶ and each P¹⁸ are each independently selected from H, C₁-C₆alkyl, and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl heteroaryl, C₁-C₆alkylaryl-Y-L-X—P(O)(OR^(X))(OR^(Y)) and -Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1 to 2 substituents selected from C₁-C₆alkyl or heterocyclyl with the proviso that at least one of P^(16,)P¹⁷ or a P¹⁸ contains a -Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety; R^(X) and R^(Y) are independently selected from H and C₁-C₆alkyl; R^(C), R^(D) and R^(H) are each independently selected from H and C₁-C₆alkyl; X^(C) is selected from CH and N; R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—; X^(E) is selected from a covalent bond, CR^(E2)R^(E3) and NR^(E4); R^(E2), R^(E3)R^(E4) are independently selected from H and C₁-C₆alkyl; X^(Hl)-X^(H2) is selected from —CR^(H2)R^(H3)—, —CR^(H2)R^(H3)-CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—, —CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—, C(O)NR^(H4)—, CR^(H2)R^(H3)S(O)₂ and —CR^(H2)═CR^(H2)—; R^(H2)R^(H3) R^(H4) are each independently selected from H, C₁-C₆alkyl and P¹⁸; X^(H3) is selected from N and CN; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C(O), S and NH; L is selected from, a covalent bond C₁-C₆alkylene, C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)—each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; m is selected from 0 or 1; each p is independently selected from 1, 2, 3, 4, 5 and 6; q is selected from 1, 2, 3 and 4; and s is selected from 0 and 1; Formula (G) is as follows:

wherein: P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, NR^(V)R^(W) and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹² is selected from H, C₁-C₆alkyl, aryl optionally substituted by —C(O)NR^(V)R^(W), and -Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹³, P¹⁴ and P¹⁵ are independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy and -Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ P¹⁵ is -Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(V) and R^(W) are independently selected from H, C₁-C₆alkyl or together with the nitrogen atom to which they are attached form a 4 to 7 remembered heterocyclic ring; X^(G) is selected from C, CH and N; represents an optional double bond, wherein X^(G) is C if is a double bond; and R^(G) is selected from H and C₁-C₆alkyl; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C(O), S and NH; L is selected from, a covalent bond C₁-C₆alkylene, C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)—each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2, 3, 4, 5 and 6 and q is selected from 1, 2, 3 and 4; Formula (I) and Formula (II) are as follows:

wherein: Z is —NH2 or —OH; X¹ is alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, carbocyclylene, substituted carbocyclylene, heterocyclylene, or substituted heterocyclylene; L¹ is a covalent bond, arylene, substituted arylene, heterocyclylene, substituted heterocyclylene, carbocyclylene, substituted carbocyclylene, —S—, —S(O)—, S(O)₂, or —O—; X² is a covalent bond, alkylene, or substituted alkylene; L² is NR^(S)—, —N(R⁵)C(O) —O—, —S—, —S(O)—, S(O)₂, or a covalent bond; R³ is H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, or substituted heterocyclylalkyl; Y¹ and Y² are each independently a covalent bond, —O—or -NR⁵-; or —Y¹—R¹ and —Y—R² are each independently —O—N═C(R⁶R⁷); R¹ and R² are each independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, substituted heterocyclylalkyl, -alkylene —C(O)—O—R⁵, -(substituted alkylene)—C(O)—O-R⁵, -alkylene—O—C(O)—R⁵, -(substituted alkylene)—O—C(O)—R⁵, -alkylene—O—C(O)—O—R⁵, or -(substituted alkylene)—O—C(O)—O—R⁵ R⁴ is H, halogen, —OH, —O-alkyl, —O-alkylene—O—C(O)—O-R⁵, —O—C(O)—O—R⁵, -SH, or -NH(R⁵); each R⁵, R⁶, and R⁷ are independently H, alkyl, substituted alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl, or substituted heterocyclylalkyl; Formula (J) is as follows:

wherein: R¹ is H, —C(O) -C₇-C₁₈alkyl or —C(O)—C₁-C₆alkyl; R² is C₇-C₁₈alkyl ; R³ is C₇-C₁₈alkyl; L₁ is -CH₂OC(O)—, -CH₂O—, -CH₂NR⁷C(O)— or -CH₂OC(O)NR⁷-; L₂ is -OC(O)—, —O—, -NR⁷C(O)— or -OC(O)NR⁷-; R⁴ is —L₃R⁵ or —L₄R⁵; R⁵ is —N(R⁷)₂, -OR⁷, —P(O)(OR⁷)₂, —C(O)OR⁷, -NR⁷C(O)L₃R⁸, -NR⁷C(O)L₄R⁸, -OL₃R⁶, —C (O)NR⁷L₃R⁸, —C(O)NR⁷L₄R⁸, —S(O)₂OR⁷, -OS(O)₂OR⁷, C₁-C₆alkyl, a C₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14 ring membered heteroaryl containing 1 to 3 heteroatoms selected from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O, S and N, wherein the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are each unsubstituted or the aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are each substituted with 1 to 3 substituents independently selected from -OR⁹, -OL₃R⁶, -OL₄R⁶, -OR⁷, and —C(O)OR⁷; L₃ is a Ci-Cioalkylene, wherein the Ci-Cioalkylene of L₃ is unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1 to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with 2 C₁-C₆alkyl groups on the same carbon atom which together, along with the carbon atom they are attached to, form a C₃-C₈cycloakyl; L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—, wherein each R¹¹ is a C₁-C₆alkyl groups which together, along with the carbon atom they are attached to, form a C₃-C₈cycloakyl; each R⁶ is independently selected from halo, C₁-C₆alkyl, C₁-C₆alkyl substituted with 1-2 hydroxyl groups, -OR⁷, —N(R⁷)₂, —C(O)OH, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀aryl and a C₁₄aryl; each R⁷ is independently selected from H and C₁-C₆alkyl; R⁸ is selected from —SR⁷, —C(O)OH, —P(O)(OR⁷)₂, and a 5 to 6 ring membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O and N; R⁹ is phenyl; each R¹⁰ is independently selected from H and halo; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4; Formula (K) is as follows:

wherein: R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, —L¹R⁵, —L¹R⁶, —L²R⁵, —L²R⁶, —OL²R⁵, or -OL²R⁶; L¹ is —C(O)— or —0—; L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p), wherein the C₁-C₆alkylene and C₂-C₆alkenylene of L² are optionally substituted with 1 to 4 fluoro groups; each L³ is independently selected from C₁-C₆alkylene and —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³ is optionally substituted with 1 to 4 fluoro groups; L⁴ is arylene or heteroarylene; R² is H or C₁-C₆alkyl; R³ is selected from C₁-C₄alkyl, —L³R⁵, —L¹R⁵, —L³R⁷, —L³L⁴L³R⁷, —L³L⁴R⁵, —L³L⁴L³R⁵, —OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH ; each R⁴ is independently selected from H and fluoro; R⁵ is —P(O)(OR⁹)₂, R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰; R⁷ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰; R⁸ is H or C₁-C₄alkyl; each R⁹ is independently selected from H and C₁-C₆alkyl; R₁₀ is H or C₁-C₄alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4; Formula (F) is as follows:

wherein: X³ is N; X⁴ is N or CR³ X⁵ is —CR⁴═CR⁵—; R¹ and R² are H; R³ is H; R⁴ and R⁵ are each independently selected from H, halogen, —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹ _(R) ¹²)_(, —N(R) ¹¹R¹²), —N(R⁹)₂, —NHN(R⁹)₂, —SR⁷, -(CH₂)_(n)OR⁷, -(CH₂)_(n)R⁷, —LR⁸, —LR¹⁰, —OLR⁸, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ and R⁵ are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, -NO₂, -R⁷, -OR⁸, —C(O)R⁸, -OC(O)R⁸, —C(O)OR⁸, -N(R⁹)₂, —P(O)(OR⁸)₂, -OP(O)(OR⁸)₂,) —P(O)(OR¹⁰)₂. -OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂, —S(O)₂R⁸, —S(O)R⁸, —S(O)₂N(R⁹)₂, and -NR⁹S(O)₂R⁸; or, R³ and R⁴, or R⁴ and R⁵, or R⁵ and R⁶, when present on adjacent ring atoms, can optionally be linked together to form a 5-6 membered ring, wherein the 5-6 membered ring is optionally substituted with R⁷; each L is independently selected from a bond, —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and C₂-C₆alkynylene of L are each optionally substituted with 1 to 4 substituents independently selected from halogen, -R⁸, -OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, -OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and -OP(O)(OR¹⁰)₂; R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl groups of R⁷ are each optionally substituted with 1 to 3 R¹³ groups, and each R¹³ is independently selected from halogen, -CN, —LR⁹, -LOR⁹, -OLR⁹, -LR¹⁰, -LOR¹⁰, -OLR¹⁰, LR⁸, -LOR⁸, OLR⁸, -LSR⁸, -LSR¹⁰, -LC(O)R⁸, -OLC(O)R⁸, -LC(O)⁸, -LC(O)R¹⁰, -LOC (O)OR⁸, -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸, -LNR⁹R¹° , -LC(O)N(R⁹)₂, -LS (O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH, -LNR⁹C(O)R⁸, -LNR⁹C(O)OR₂, -LS(O)₂N(R⁹)₂, -OLS (O)₂N(R⁹)₂, -LNR⁹S(O)₂R⁸, -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂, -LP (O)(OR¹⁰)₂ and -OLP(O)(OR¹⁰; each R⁸ is independently selected from H, -CH(R¹⁰)₂, C₁-C₈alkyl, C₂-C₈alkene, C₂-c₈alkyne, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and C₁-C₆haloalkoxy groups of R⁸ are each optionally substituted with 1 to 3 substituents independently selected from -CN, R¹¹, -OR¹¹, -SR¹¹, -C (O)R¹¹, -OC(O)R¹¹, —C(O)N(R⁹)₂, —C(O)OR¹¹, -NR⁹C(O)R¹¹, -NR⁹R¹⁰, -NR¹¹R¹², -N(R⁹)₂, OR⁹, -OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹, —S(O₂NR¹¹R¹², -NR¹¹S(O₂R¹¹, —P(O)(OR¹¹)₂, and -OP(O)(OR¹¹)₂; each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸, —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, -C₁-C₆ alkyl, C₁-C₆ heteroalkyl and C₃-C₆ cycloalkyl, or each R⁹ is independently a C₁-C₆alkyl that together with N they are attached to form a C₃-C₈heterocycloalkyl, wherein the C₃-C₈heterocycloalkyl ring optionally contains an additional heteroatom selected from N, O and S, and wherein the C₁-C₆ alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or C₃-C₈heterocycloalkyl groups of R⁹ are each optionally substituted with 1 to 3 substituents independently selected from -CN, R¹¹, -OR¹¹, -SR¹¹, —C(O)R¹¹, OC(O)R¹¹, —C(O)OR¹¹, -NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)2R¹¹, —S(O)R¹¹, —S(O)₂NR¹¹R¹², -NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂ and -OP (O)(OR¹¹)₂; each R¹⁰ is independently selected from aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl, wherein the aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl groups are optionally substituted with 1 to 3 substituents selected from halogen, —R⁸, -OR⁸, -LR⁹, -LOR⁹, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸. —C(O)₂R⁸, —C(O)R⁸ and —C(O)N(R⁹)₂; R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆aloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the C₁-C₆aIkyl, C ₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R¹¹ and R¹² are each optionally substituted with 1 to 3 substituents independently selected from halogen, -CN, R⁸, -OR⁸, C(O)R⁸, OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, -NR ⁸C(O)R⁸, -NR⁸C(O)OR⁸, —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂, —S(O)₂N(R⁹)₂, -NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and C₁-C₆haloalkoxy; or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken together with the N atom to which they are attached form an optionally substituted C₃-C₈heterocycloalkyl ring optionally containing an additional heteroatom selected from N, O and S; ring A is an aryl or a heteroaryl, wherein the aryl and heteroaryl groups of Ring A are optionally substituted with 1 to 3 R^(A) groups, wherein each R^(A) is independently selected from -R⁸, -R7, -OR⁷, -OR⁸, -R¹⁰, -OR¹⁰, SR⁸, —NO₂, -CN, —N(R⁹)₂, -NR⁹C(O)R⁸, -NR⁹C(S)R⁸, -NR⁹C(O)N(R⁹)₂, - NR⁹C(S)N(R⁹)₂, -NR⁹CO₂R⁸, -NR⁹NR⁹C(O)R⁸, -NR⁹NR⁹C(O)N(R⁹)₂, -NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸, -CO₂R⁸, -(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂, -OC(O)N(R⁹)₂, -OC(O)R⁸, —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸, —S(O)₃R⁸, -SO₂N(R⁹)₂, —S(O)R⁸, -NR⁹SO₂N(R⁹)₂, -NR⁹SO₂R⁸, —P(O)(OR⁸)₂, -OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, -OP(O)(OR¹⁰)₂, —N(OR⁸)R⁸, -CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and -(CH₂)_(n)NHC(O)R⁸ or two adjacent R^(A) substituents on Ring A form a 5-6 membered ring that contains up to two heteroatoms as ring members; n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6, 7 or 8; each m is independently selected from 1, 2, 3, 4, 5 and 6; and t is 1, 2, 3, 4, 5, 6, 7 or
 8. 12. The immunogenic composition of claim 1, further comprising a histidine buffer.
 13. The immunogenic composition of claim 1, with a diphtheria toxoid concentration ≤4 Lf/ml.
 14. The immunogenic composition of claim 1, with a tetanus toxoid concentration ≤9 Lf/ml.
 15. The immunogenic composition of claim 1, with a pertussis toxoid concentration ≤4 μg/ml.
 16. The immunogenic composition of claim 1, further comprising a trivalent inactivated poliovirus antigen component.
 17. The immunogenic composition of claim 1, having a pH between 6.1 and 7.9.
 18. A method of raising an immune response in a subject, comprising the step of administering to the subject the composition of claim
 1. 19. The method of claim 18, wherein the subject has previously received a DTP vaccine as a child.
 20. An immunogenic composition comprising a diphtheria toxoid, a tetanus toxoid, a pertussis toxoid, and an oil-in-water emulsion adjuvant, wherein the immunogenic composition comprises an excess (measured in Lf units) of tetanus toxoid relative to diphtheria toxoid.
 21. The immunogenic composition of claim 20, wherein the emulsion comprises squalene and/or polysorbate
 80. 22. The immunogenic composition of claim 20, wherein at least 80% by number of oil droplets in the emulsion have a diameter of less than 220 nm.
 23. The immunogenic composition of claim 20, with a diphtheria toxoid concentration ≤4 Lf/ml.
 24. The immunogenic composition of claim 20, with a tetanus toxoid concentration ≤9 Lf/ml.
 25. The immunogenic composition of claim 20, with a pertussis toxoid concentration ≤4 μg/ml.
 26. The immunogenic composition of claim 20, further comprising a trivalent inactivated poliovirus antigen component.
 27. The immunogenic composition of claim 20, having a pH between 6.1 and 7.9.
 28. A method of raising an immune response in a subject, comprising the step of administering to the subject the immunogenic composition of claim
 21. 29. The method of claim 28, wherein the subject has previously received a DTP vaccine as a child.
 30. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, wherein the pertussis toxoid is the PT-9K/129G double mutant.
 31. The immunogenic composition of claim 30, in unit dose form, and comprising 4 μg pertussis toxoid, 4 μg FHA and 8 μg pertactin per unit dose.
 32. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, the immunogenic composition further comprising diphtheria toxoid and tetanus toxoid.
 33. An immunogenic composition comprising an acellular pertussis component in which a pertussis toxoid, filamentous hemagglutinin and pertactin are present at a mass ratio of 1:1:2, the immunogenic composition further comprising a TLR agonist adsorbed to an aluminium salt adjuvant. 